Journal of Nuclear Medicine,45, 216P (2004)

Transport of [¹¹C]meta-hydroxyephedrine, [¹¹C]epinephrine, and biogenic amines by the human norepinephrine transporter.

David M. Raffel,¹ W. Chen,¹ David L. Gildersleeve,¹ Yong-Woon Jung,¹ and Nicholas V. Cozzi^2,3

¹Division of Nuclear Medicine, Dept. of Radiology, University of Michigan Medical School, Ann Arbor, MI

²Department of Pharmacology and Toxicology, Brody School of Medicine, East Carolina University, Greenville, NC 27834

³Present address: PhysioGenix, Inc., 10437 Innovation Drive, Wauwatosa, WI 53226

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Abstract

Objectives: [¹¹C]meta-hydroxyephedrine (HED) and [¹¹C]epinephrine (EPI) are used to assess cardiac sympathetic nerve integrity with PET. Like the endogenous neurotransmitter norepinephrine, HED and EPI are transported into sympathetic neurons by the norepinephrine transporter (NET) and stored in vesicles. To better characterize the kinetics of HED and EPI, their Michaelis-Menten transport parameters K_m and V_max for NET transport were measured in vitro using rat C6 glial cells that stably express the human NET (C6-hNET cells). Transport parameters for [³H]norepinephrine (NE) and [³H]dopamine (DA) were also measured. Methods: C6-hNET cells were grown to confluency in 24-well plates. Triplicate measures of total and nonspecific HED uptake were made by incubating cells for 5 min with varying HED concentrations (0.1 – 5 uM; 37 ºC). Nonspecific uptake was measured using 10 uM desipramine to block NET transport. After rinsing, cells were solubilized and counted in a gamma counter. K_m and V_max were estimated by fitting specific uptake vs. HED concentration to a one-site transport model. Similar methods were used for EPI, NE and DA, using liquid scintillation counting for [³H]. Results: Transport parameters are shown in the Table. The ratio V_max/K_m gives each substrate’s NET transport rate. HED is transported by hNET at half the rate of NE. EPI, with a K_m more than 10 times higher than NE, is transported at only one-tenth the rate of NE and one-fifth the rate of HED. Conclusion: Since V_max is directly proportional to NET density, estimates of V_max/K_m from a tracer’s PET kinetics would provide sensitive measures of regional sympathetic nerve density. HED and EPI exhibit very similar in vivo kinetics, despite a five-fold different NET transport rate. Kinetic analyses of HED and EPI fail because their neuronal uptake rates (i.e., V_max/K_m ratios) are too fast. A new sympathetic nerve tracer must have a V_max/K_m ratio lower than EPI’s for its in vivo kinetics to be suitable for tracer kinetic analyses.

Substrate	Km (uM)	Vmax (nmol/min/mg protein)	Vmax/Km
HED	0.49 ± 0.11	5.20 ± 0.90	10.7 ± 0.9
NE	0.28 ± 0.03	5.83 ± 0.49	21.3 ± 2.4
EPI	3.16 ± 0.78	6.09 ± 1.04	2.0 ± 0.2
DA	0.24 ± 0.04	2.91 ± 0.47	12.2 ± 1.8

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